Salmonella enter epithelial cells during infection. In order to elucidate how Salmonella enter these non-phagocytic cells, bacterial genes have been identified that are required for bacterial entry. This proposal focuses on the hyperinvasion locus (hil) of S. typhimurium which encodes essential invasion genes. The hil genes map in a region of the S. typhimurium chromosome that encodes many other genes required for invasion. It is likely that, together, hil and the adjacent invasion genes encode factors required for the expression, assembly and composition of an invasion structure on the surface of S. typhimurium. The basic goals of the proposal are 1) to understand the role of hil in invasion and 2) to examine the importance of hil and bacterial invasion of S. typhimurium pathogenicity. Specifically, we plan to study 1) the hil genes and gene products 2) the interaction of hil mutants with epithelial cells in vitro and in vivo 3) the effect of hil mutations on S. typhimurium pathogenicity. A combination of experimental techniques will be used, including bacterial genetics, molecular biology, in vitro infection models in which S. typhimurium enter cultured epithelial cells, and in vivo murine infection models. Completion of the experiments will lead to the genetic and biological characterization of Salmonella invasion factors. In addition, the role of these bacterial factors in epithelial cell entry and pathogenicity will be determined. Salmonella remains a persistent national and worldwide health problem. The most likely candidate for an effective vaccine is a live attenuated Salmonella strain which can infect the intestinal mucosa and stimulate a protective immune response. The long-term objective of this proposal is to understand the interactions of Salmonella with intestinal epithelial cells. These studies may have direct application in vaccine development.